MEMBRANE PROTEIN TOPOLOGY--CYTOCHROME C

细胞膜结构与功能的阐明 今天，科学仍然是科学的重大挑战之一。 的 当我们试图了解分子基础时， 这种常见的致命遗传疾病，如囊性纤维化， 缺陷归因于跨膜氯离子通道故障。 通道是如何构建在膜中的？膜是否影响 渠道活动？它与哪些关键组件相互作用， 在膜的旁边这些都是关于膜的关键问题 蛋白质拓扑学由于缺乏适当的方法而仍然没有答案 和模型系统。 考虑到这些长远目标，我们建议 为了解决膜拓扑结构挑战的特定方面， 引入一种新的实验方法。 这将用于确定 细胞色素c的精确分布， 运输系统，在膜表面。 虽然晶体结构 蛋白质的已知，细胞色素c停靠在蛋白质上的方式 膜表面，以及它如何在功能上与它的伙伴相互作用， 电子传输系统仍然是个问题。 X射线驻波 (XSW)将首次用于确定亚埃 分辨率，固有重原子铁的位置，在一个 细胞色素c单层静电结合于Langmuir-Blodgett 沉积在固体支持物上的脂质膜。 后者将用于 生成XSW。 蛋白质方向将通过执行以下操作来确认： 额外的XSW测量以确定i）硒原子在 用硒代甲硫氨酸在三个或更多个位点修饰的细胞色素c 取代天然甲硫氨酸和ii）钌原子在 细胞色素c在蛋白质表面上的Cysl 02处单独衍生。 在 通过这种方式，可以确定至少五个已知参考点的位置， 将测定膜表面以上的蛋白质。 连同 与已知的蛋白质结构，将被用来建立 细胞色素c在膜表面的取向是明确的。 将测定还原和氧化后的取向变化 并与晶体结构中观察到的构象变化相关。 此外，蛋白质在水相中的分布接下来 以及膜脂是如何调节的 特性以及膜浴的离子组成 解决方案将被确定，并用于测试假设，至少 线粒体中存在两个细胞色素C库。 单独测量 将利用脱辅基细胞色素c和细胞色素c共价结合， 膜表面。 膜对接的成功测定 细胞色素c的机制以及蛋白质 分布在膜结合和可溶性形式之间， 用XSW技术阐明了 一系列的整合和外周蛋白质，其膜特性 由于缺乏适当的方法，仍然难以实现。

The elucidation of the structure and function of cellular membrane components remains today one of Science's grand challenges. The challenge becomes immediate as we seek to understand the molecular basis of such common lethal genetic diseases as cystic fibrosis where the defect is attributed to a malfunctioning transmembranal chloride channel. How is the channel built into the membrane? Does the membrane influence channel activity? What critical components does it interact with in, on, and next to the membrane? These are crucial questions concerning membrane protein topology that remain unanswered for want of appropriate methods and model systems. With these long term objectives in mind, we propose to tackle particular aspects of the membrane topology challenge by introducing a new experimental approach. This will be used to determine the precise disposition of cytochrome c, a component of the electron transport system, at the membrane surface. While the crystal structure of the protein is known, the manner in which cytochrome c docks on the membrane surface and how it interacts functionally with its partners in the electron transport system is still in question. X-ray standing waves (XSW) will be used for the first time to determine, with subangstrom resolution, the position of the intrinsic heavy atom, iron, In a cytochrome c monolayer electrostatically bound to a Langmuir-Blodgett lipid film deposited on a solid support. The latter will be used to generate the XSWs. Protein orientation will be confirmed by performing additional XSW measurements to determine i) selenium atom location in cytochrome c modified at three or more sites with selenomethionine substituting for the native methionine and ii) ruthenium atom location in cytochrome c derivatized singly at Cysl02 on the protein surface. In this way, the location of at least five known reference points in the protein above the membrane surface will be determined. This, together with the known structure of the protein, will be used to establish unequivocally the orientation of cytochrome c at the membrane surface. Changes in orientation upon reduction and oxidation will be determined and correlated with conformational changes seen in the crystal structure. Additionally, the distribution of the protein in the aqueous phase next to the membrane and how this is modulated by the membrane lipid characteristics as well as the ionic composition of the membrane bathing solution will be determined and used to test the hypothesis that at least two pools of cytochrome c exist in mitochondria. Separate measurements will make use of the apocytochrome c and cytochrome c covalently bound to the membrane surface. A successful determination of the membrane docking mechanism for cytochrome c and the manner in which the protein distributes between membrane-bound and soluble forms paves the way for the elucidation by the XSW technique of the structure and disposition of a host of integral and peripheral proteins whose membrane properties remain elusive for lack of appropriate methodologies.